Anastasia Kyriakoudi

Revisiting extraction of bioactive apocarotenoids from Crocus sativus L. dry stigmas (saffron)

An ultrasound assisted extraction method is proposed for the recovery of bioactive glycosides (i.e. crocins and picrocrocin) from Crocus sativus L. dry stigmas using aqueous methanol. Response surface methodology (RSM) was employed to optimize the extraction parameters, namely, the percentage of methanol (%), the duration (min) and the duty cycles (s) of sonication. Optical microscopy, spectrophotometry and RP-HPLC-DAD were employed to follow pros and cons of the process. Additional experiments were conducted to compare recoveries with those under other agitation conditions (e.g. magnetic stirring according to ISO 3632-2 standard). The percentage of methanol, the sonication duration and duty cycles combination that can be recommended as optimum for the recovery of crocins and picrocrocin were 50%, 30 min, 0.2s and 0.44%, 30 min, 0.6s, respectively. Picrocrocin levels were not influenced dramatically under the optimum conditions for crocins extraction (11±2 instead of 12±1 mg kg(-1) dry stigmas, respectively) so that these can be considered optimum for both categories of tested compounds. Ultrasound assisted extraction speeded up further recovery of these precious apocarotenoids. Our findings for extraction conditions are useful for both industrial and analytical applications and should be considered in a forthcoming revision of the ISO 3632-2 technical standard.

Changes in Total and Individual Crocetin Esters upon in Vitro Gastrointestinal Digestion of Saffron Aqueous Extracts

Changes that may be expected in crocetin esters (crocins) upon digestion were examined in saffron aqueous extracts for the first time. Chemical characterization of total and individual crocins and other bioactive compounds was achieved by UV-vis spectrophotometry, RP-HPLC-DAD, and LC-ESI-MS. Antioxidant activity was evaluated using in vitro assays and the comet assay. The observed loss for both total and trans-crocins was higher in saffron (∼50%) than in gardenia extracts (∼30%), which were also examined for comparison. Loss was lower than that reported for hydrophobic carotenoids. cis-Isomers were less affected, leading to the hypothesis that trans/cis isomerization may occur in parallel to degradation reactions. Monitoring changes in the extracts at oral, gastric, or intestinal phases, separately, verified this view pointing out the critical effect of pH, temperature, and duration of process but not of digestive enzymes. No isomerization and less degradation (<20% loss) was evidenced when pure trans-crocetin (di-β-D-gentiobiosyl) ester was subjected to gastric or intestinal conditions.

Cellular Transport and Bioactivity of a Major Saffron Apocarotenoid, Picrocrocin (4-(β-d-Glucopyranosyloxy)-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde)

The cellular transport and bioactivity of the second major saffron apocarotenoid, picrocrocin, was examined in parallel to that of the major group, crocetin sugar esters, in aqueous extracts. The transport of pure picrocrocin was investigated in comparison to that of other saffron apocarotenoids, trans-crocetin (di-β-D-gentiobiosyl) ester and crocetin using the Caco-2 cell model coupled with an in vitro digestion procedure. RP-HPLC-DAD was employed to quantify the bioaccessible and bioavailable amounts of individual apocarotenoids. Picrocrocin and crocetin sugar esters though highly bioaccessible (75% and 60%, respectively) were transported at minute quantities (0.2% and 0.5%, respectively; 10-fold lower than crocetin). Picrocrocin did not protect against oxidant-induced DNA damage in U937, human monocytic blood cells at the concentration investigated, however, it reduced the proliferation of human adenocarcinoma and hepatocarcinoma cells. Our findings may be useful for the requirements of food legislation regarding saffron preparations, in which both apocarotenoid groups coexist.

Enhanced Bioaccessibility of Crocetin Sugar Esters from Saffron in Infusions Rich in Natural Phenolic Antioxidants

The present study aims to examine whether and to what extent the bioaccessibility of the major saffron apocarotenoids, namely crocetin sugar esters (CRTSEs), is affected by the presence of strong water-soluble antioxidants, ingredients of the herbs found in commercial tea blends with saffron. An in vitro digestion model was applied to infusions from these products to investigate the possible changes. All of the studied infusions were rich in total phenols (9.9–22.5 mg caffeic acid equivalents/100 mg dry infusion) and presented strong DPPH radical scavenging activity regardless of the composition of the corresponding herbal blends. RP-HPLC-DAD and LC-MS analysis enabled the grouping of the infusions into hydroxycinnamic acid-rich and in flavan-3-ol-rich ones. CRTSEs in herbal tea infusions were found to be significantly more bioaccessible (66.3%–88.6%) than those in the reference saffron infusion (60.9%). The positive role of strong phenolic antioxidants (caffeic acid, rosmarinic acid) on the stability of CRTSEs was also evidenced in model binary mixtures. On the contrary, cinnamic acid, exerting no antioxidant activity, did not have such an effect. Our findings suggest that strong radical scavengers may protect the crocetin sugar esters from oxidation during digestion when present in excess.

Sensory Threshold Studies of Picrocrocin, the Major Bitter Compound of Saffron

This study is part of a wider project on the bitter taste of saffron and its preparations. A deeper knowledge on the taste perception of picrocrocin is necessary in order to develop products that satisfy consumer senses and provide them with adequate amounts of saffron major constituents, also appreciated for bioactivity. A systematic approach on the bitterness of picrocrocin, the major responsible compound, was conducted. A panel was trained specifically for the determination of taste detection and recognition thresholds of picrocrocin, which were found to be 5.34 and 7.26 mg/L, respectively, using the Ascending Forced Choice of Limits methodology. The threshold values were examined in water in absence and presence of other saffron constituents and ethanol and were found to decrease when served hot (61 ± 4 °C). Bitterness was enhanced in 40% (v/v) aqueous ethanol. In both aqueous and ethanol extracts, the presence of saffron volatiles improved bitterness perception. The usefulness of the study was tested in the case of commercial saffron based infusions.

Calpain-Dependent Death in C6 Rat Glioma Cells, Exhibiting a Synergistic Effect with Temozolomide

Abstract Crocus sativus L., a dietary herb, has been used for various diseases including cancer. This is an in vitro study investigating the antineoplastic effect of the extract of the plant against C6 glioma rat cell line. The mechanism of cellular death and the synergistic effect of the extract with the alkylating agent temozolomide (TMZ) were investigated. Cellular viability was examined in various concentrations of the extract alone or in combination with TMZ. Apoptosis was determined with flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and autophagy by western blotting of the light chain 3 (LC3)-II. Cellular viability was reduced after exposure to the extract with half maximal inhibition concentration at 3 mg/ml. Flow cytometry and TUNEL assay suggested that the extract does not induce apoptosis. Moreover, their combination increased the ratio dead/apoptotic cells 10-fold (P < 0.001). LC3-II protein levels reduced after Crocus extract while this effect was reversed when the calpain inhibitor MDL28170 was added, suggesting a calpain-dependent death possibly through autophagy. We concluded that the extract of Crocus increases dead cell number after 48 h of exposure. Our results suggest that the cell undergoes calpain-dependent programmed cell death while co-exposure to Crocus extract and TMZ enhances the antineoplastic effect of the latter.

Properties of encapsulated saffron extracts in maltodextrin using the Büchi B-90 nano spray-dryer

The production, characterization and stability of nanoencapsulates of saffron hydrophilic apocarotenoids, i.e. crocins and picrocrocin, in maltodextrin using spray-drying are presented. The effect of mesh size and core:wall ratio (w/w) on the product yield and encapsulation efficiency of these apocarotenoids was examined. Nanoencapsulates were characterized and their stability was examined in the presence or absence of a strong phenolic antioxidant, the caffeic acid, under thermal and in vitro gastrointestinal conditions. Spherical particles were obtained. Product yield and encapsulation efficiency (%) of crocins and picrocrocin was found to be satisfactory. Thermal stability and bioaccessibility of these apocarotenoids was enhanced by nanoencapsulation. Further protection was provided by caffeid acid.

Latest advances in the extraction and determination of saffron apocarotenoids

Saffron, the dried red stigmas of Crocus sativus L. plant, is the most expensive spice in the world. It is highly valued not only for the color and flavor that exerts to various foods and drinks but also for its functional properties. Various classes of apocarotenoids such as crocetin sugar esters, picrocrocin and safranal are responsible for these characteristics. In the present review, the evolution of the methods proposed by the ISO standard for extraction and determination of saffron apocarotenoids since 1980 is presented in parallel to other approaches proposed by various scientists to overcome limitations of the standard. Moreover, the latest advances regarding applications of novel extraction techniques and powerful analytical tools that require limited or no sample preparation are critically discussed.

A stepwise approach for the detection of carminic acid in saffron with regard to religious food certification

The stepwise approach takes advantage of simple, versatile, low-cost screening tools that can be applied to several posts of the saffron trade chain to specifically detect adulteration with carminic acid (CA). This natural dye is of insect origin and should not be present in Kosher and Halal foods such as saffron. For gross adulteration levels (>25.0%, w/w) reaction with diphenylamine-sulfuric acid was found adequate to indicate the presence of extraneous matter but not its identity. FT-IR analysis of the dry material combined with chemometrics served to rapidly sort out samples containing >10.0% CA without any sample pretreatment except grinding. Aqueous extracts prepared according to ISO 3632-2 were then examined by tristimulus colorimetry and derivative UV-Vis spectrometry to detect adulteration down to the level of 2.0% (w/w). Determination of CA down to 0.2%, w/w was achieved by RP-HPLC-DAD using aqueous acetonitrile elution solvent (pH=2.8).